The overall objective in this past year was to validate and select hits from the previously completed qHTS campaign for medicinal chemistry probe development. During this period, the team found that, out of compounds from 8 chemotypes and 6 singletons, only one hit (surfen) showed consistent activity in our panel of primary, orthogonal and selectivity assays upon purification. A novel high-throughput cellular thermal shift assay (CETSA) using a split NanoLuciferase (NanoLuc) was developed for bacterial applications. Sufen displayed a 2 degree C shift in the CETSA assay, suggesting possible target-binding to VapC-1. The first crystal structure of VapBC had also been successfully obtained. Additionally, a virtual screening for VapC inhibitors was conducted, and more than 40 virtual screening hits' activity has been confirmed in the primary assay.